This application relates to serine protease inhibitors, inhibitor-enzyme complexes formed by the serine protease inhibitors, methods of using the serine protease inhibitors and the inhibitor-enzyme complexes, and methods for determining whether compounds inhibit serine proteases.
Serine proteases are produced within the cells of many living organisms and are often secreted to act outside of the producing cell. Individual serine proteases may target specific substrates (e.g., an inactive precursor for conversion to its biologically active form) or may act non-specifically (e.g., degradation of proteins or other peptides by scission). Further, individual serine proteases may be highly selective in that they recognize only one or a few related subsequences or non-selective in that they recognize and cleave a variety of unrelated sequences.
Serine proteases have a highly conserved active site, wherein specific amino acids which catalyze the bond scission have nearly identical spatial arrangements. A complementary binding site adjacent to the active site provides for the primary specificity of any individual serine protease. A succession of indentations or xe2x80x9cpocketsxe2x80x9d along the surface of the protease serve to bind successive amino acid side chains along the substrate polypeptide chain on either side of the peptide bond to be cleaved. Substrate side chains which contribute to the association with the protease are designated P1, P2, P3, etc., proceeding from the side chain proximate to the susceptible bond toward the amino terminal of the protein, and P1xe2x80x2, P2xe2x80x2, P3xe2x80x2, etc., proceeding from the side chain proximate to the susceptible bond toward the carboxyl terminal of the protein. Small molecules having suitable P binding moieties can be designed to mimic the substrate by occupying the substrate""s binding site and inhibit the function of the serine protease.
Serine proteases provide a diverse array of biological functions. Important serine proteases include trypsin-like proteases, such as trypsin, tryptase, thrombin, plasma kallikrein, tissue kallikrein and factor Xa. Substrates for serine proteases are associated, for example, with blood clotting, complement mediated lysis, the immune response, glomerulonephritis, pain sensing, inflammation, pancreatitis, cancer, regulating fertilization, bacterial infection and viral maturation. Accordingly, appropriate drug therapies can comprise the inhibition of a particular serine protease implicated in the pathology and/or symptomatology of a disease. Hence, substantial interest exists in the identification of serine protease inhibitors which possess high selectivity for specific serine proteases.
The disclosure of other documents referred to throughout this application are incorporated herein by reference.
An aspect of this invention is a method for determining the serine protease inhibitory activity of a compound, which method comprises contacting the compound with a serine protease in a medium having present therein a divalent metal cation, wherein the cation has the capacity for interaction with the compound and thereby to potentiate any serine protease inhibitory activity possessed by the compound and the concentration of the divalent metal cation in the medium is modified to a level sufficient to produce any such interaction.
A second aspect of this invention is a method for determining whether the inhibitory activity of a serine protease inhibitor is potentiated by the presence of a divalent metal cation, which method comprises:
(a) assaying for the inhibition of a serine protease by the inhibitor, wherein the assay is conducted in a medium that is essentially devoid of dissociated divalent metal cations; and
(b) assaying for the inhibition of the serine protease by the compound under essentially equivalent assay conditions to those used in Step (a), with the exception that the assay performed in Step (b) is conducted in a medium that contains an effective concentration of a divalent metal cation; wherein the inhibitory activity of the compound when measured by Step (b) is significantly greater than the inhibitory activity of the compound when measured by Step (a).
A third aspect of this invention is a method for inhibiting a serine protease with a serine protease inhibitor in a medium comprising the serine protease and the inhibitor, in which the inhibitor comprises two heteroatoms in spatial relationship one to the other so as to chelate a physiologically acceptable divalent metal cation capable of chelation, the improvement which comprises having sufficient quantity of the divalent metal cation in the medium or adding a sufficient amount of the divalent metal cation to the medium to have any or all of the inhibitor which is bound to the serine protease as a divalent metal cation complex, or providing the inhibitor as a divalent metal cation binary complex, such that the divalent metal cation is bound between the inhibitor and the serine protease as a divalent metal cation ternary complex.
A fourth aspect of this invention is a divalent metal cation binary complex of a compound of Formula I:
{(BP)rxe2x80x94(I)}n(Y)qxe2x80x83xe2x80x83I
in which:
q is 0 and n is 1 or
q is 1 and n is 2;
Y is a bond or linking group of not more than six, typically not more than three and preferably carbon, atoms in a chain;
BP is a binding moiety for binding to one or more P sites of a serine protease;
r is 0 or 1, with the proviso that at least one BP binding moiety is present; and
I is a moiety that comprises at least one heteroatom when n is 2 and comprises at least two heteroatoms when n is 1, wherein two heteroatoms are in spatial relationship one to the other so as to be able to chelate the divalent metal cation in a bidentate manner.
A fifth aspect of this invention is a divalent metal cation ternary complex comprising the compound of Formula I in association with the divalent metal cation and a serine protease.
A sixth aspect of this invention is the serine protease inhibitors identified by the methods of this invention.
A seventh aspect of this invention is a method for treating a disease in an animal in which serine protease activity contributes to the pathology and/or symptomatology of the disease, which method comprises administering to the animal a therapeutically effective amount of a serine protease inhibitor identified by the methods of this invention.
Thus, it is intended that the scope of this invention encompasses any method for determining the serine protease inhibitory activity of a compound wherein the method requires the presence of a divalent metal cation and a serine protease and one of ordinary skill in the art could demonstrate that the cation has the capacity for interaction with a compound of Formula I and thereby to potentiate the inhibitory activity of the compound or is capable of chelating simultaneously with a compound of Formula I and the active site of the serine protease and thereby potentiate the inhibitory activity of the compound.
Definitions
Unless otherwise stated, the following terms used in the specification and claims have the meanings given below:
xe2x80x9cAlkylxe2x80x9d means a straight or branched, saturated or unsaturated hydrocarbon radical having the number of carbon atoms indicated (e.g., (C1-8)alkyl includes methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, tert-butyl, vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, 3-butenyl, 2-methylallyl, ethynyl, 1-propynyl, 2-propynyl, etc.).
xe2x80x9cAlkylenexe2x80x9d means a straight, saturated or unsaturated hydrocarbon divalent radical (e.g., methylene, ethylene, vinylene, ethynylene, 2-propylene, 1-propylene, tetramethylene, isopropylidene, etc.).
xe2x80x9cAmidinoxe2x80x9d means the radical xe2x80x94C(NH)NH2.
xe2x80x9cAminoxe2x80x9d means the radical xe2x80x94NH2.
xe2x80x9cArylxe2x80x9d means an aromatic monocyclic or polycyclic hydrocarbon radical containing the number of carbon atoms indicated, wherein the carbon atom with the free valence is a member of an aromatic ring (e.g., (C6-14)aryl includes phenyl, naphthyl, anthracenyl, phenanthrenyl, 1,2,3,4-tetrahydronaphth-5-yl, etc.).
xe2x80x9cArylenexe2x80x9d means an aromatic monocyclic or polycyclic hydrocarbon divalent radical containing the number of carbon atoms indicated, wherein the carbon atoms with the free valence are members of an aromatic ring (e.g., (C6-14)arylene includes 1,2-phenylene, 1,3-phenylene, 1,2-naphthylene, 1,3-naphthylene, 1,3-anthracenylene, 1,3-anthracenylene, 1,2-phenanthrenylene, 1,2,3,4-tetrahydro-5,6-naphthylene, etc.)
xe2x80x9cCycloalkylxe2x80x9d means a saturated or unsaturated, monocyclic or polycyclic hydrocarbon radical containing the number of carbon atoms indicated, wherein the carbon atom with the free valence is a member of a non-aromatic ring (e.g., (C3-14)cycloalkyl includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, 2,5-cyclohexadienyl, bicyclo[2.2.2]octyl, 1,2,3,4-tetrahydronaphth-1-yl, etc.).
xe2x80x9cCycloalkylenexe2x80x9d means a saturated or unsaturated, monocyclic or polycyclic hydrocarbon divalent radical containing the number of carbon atoms indicated, wherein the carbon atoms with the free valence are members of a non-aromatic ring (e.g., (C3-6)cycloalkylene includes 1,2-cyclopropylene, 1,2-cyclobutylene, 1,3-cyclobutylene, 1,2-cyclopentylene, 1,3-cyclopentylene, 1,2-cyclohexylene, 3-cyclohexen-1,2-ylene, 2,5-cyclohexadien-1,3-ylene, 1,2-bicyclo[2.2.2]octylene, 1,2,3,4-tetrahydro-1,2-naphthylene, etc.).
xe2x80x9cEssentially devoid of dissociated divalent metal cationsxe2x80x9d, as in a medium essentially devoid of dissociated divalent metal cations, means that the free concentration of the cation in the medium is so low as to have no or minimal capability of chelating with the prospective serine protease inhibitor.
xe2x80x9cEffective concentrationxe2x80x9d, as in a medium containing a compound, a serine protease and an effective concentration of divalent metal cation, means that the free concentration of cation in the medium is sufficient to produce an interaction with the compound, wherein the cation has the capacity for such interaction and thereby to potentiate any inhibitory activity of the compound, or to produce a simultaneous chelation with the compound and the active site of the serine protease, wherein the cation has the capacity for such chelation and thereby to potentiate the inhibitory activity of the compound.
xe2x80x9cFree concentrationxe2x80x9d, as in free concentration of divalent metal cation in a medium containing a compound and a serine protease, means that concentration of the cation of cation in the medium which is dissociated and free to interact with the compound, wherein the cation has the capacity for such interaction and thereby to potentiate any inhibitory activity of the compound, or to produce a simultaneous chelation with the compound and the active site of the serine protease, wherein the cation has the capacity for such chelation and thereby to potentiate the inhibitory activity of the compound.
xe2x80x9cGuanidinoxe2x80x9d means the radical xe2x80x94NHC(NH)NH2.
xe2x80x9cHeteroalkylenexe2x80x9d means alkylene, as defined above, wherein 1 to 5 of the carbon atoms indicated is replaced by a heteroatom chosen from N, O or S (e.g., amino, oxy, thio, azaethylene (xe2x80x94NHCH2xe2x80x94), oxaethylene (xe2x80x94OCH2xe2x80x94), etc.), with the proviso that the oxygen, nitrogen and sulfur atoms contained therein do not form bonds with other heteroatoms.
xe2x80x9cHeteroarylxe2x80x9d means aryl, as defined above, wherein 1 to 5 of the carbon atoms indicated are replaced by a heteroatom chosen from N, O or S.
xe2x80x9cHeteroarylenexe2x80x9d means arylene, as defined above, wherein 1 to 5 of the carbon atoms indicated are replaced by a heteroatom chosen from N, O or S.
xe2x80x9cHeteroatomxe2x80x9d means nitrogen (N), oxygen (O) or sulfur (S).
xe2x80x9cHeteroatom containing moietyxe2x80x9d, for the purposes of this application, means xe2x80x94OR, xe2x80x94NRR or xe2x80x94SR, wherein each R is independently alkyl, heteroalkyl, cycloalkyl, aryl, heterocycloalkyl, heteroaryl (e.g., methoxy, amino, methylamino, amidino, guanidino, anilino, hydroxy, mercapto, carboxy, methylacetoxy, glycinamido, cyclohexylamino, and the like.
xe2x80x9cHeterocycloalkylxe2x80x9d for the purposes of this invention means an aromatic or non-aromatic, saturated or unsaturated, monocyclic or polycyclic, fused or non-fused, hydrocarbon radical containing at least one heteroatom and the number of carbon atoms indicated (e.g., pyrazole, imidazole, triazole, oxazole, thiazole, isoxazole, benzimidazole, pyran, pyridine, pyridazine, pyrimidine, pyrazine, dioxane, quinoline, isoquinoline, cinnoline, 2,2xe2x80x2-bis-imidazole, 2,2xe2x80x2-bis-pyridine, etc.).and comprised of four to seven, typically five to six, annular members and one to four, typically one to three and more preferably one to two, annular heteroatoms.
xe2x80x9cPathologyxe2x80x9d of a disease means the essential nature, causes and development of the disease as well as the structural and functional changes that result from the disease processes.
xe2x80x9cPharmaceutically acceptablexe2x80x9d means that which is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable and includes that which is acceptable for veterinary use as well as human pharmaceutical use.
xe2x80x9cPharmaceutically acceptable saltsxe2x80x9d means salts which are pharmaceutically acceptable, as defined above, and which possess the desired pharmacological activity. Such salts include acid addition salts formed with inorganic acids such as hydrobromic acid, hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid and the like; or with organic acids such as acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, p-chlorobenzene-sulfonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, 1,2-ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hexanoic acid, heptanoic acid, o-(4-hydroxybenzoyl)benzoic acid, 2-hydroxyethanesulfonic acid, hydroxynaphthoic acid, lactic acid, lauryl sulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, 4,4xe2x80x2-methylenebis(3-hydroxy-2-ene- 1-carboxylic acid), muconic acid, 2-naphthalenesulfonic acid, oxalic acid, 3-phenylpropionic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tertiary butylacetic acid, p-toluenesulfonic acid, trimethylacetic acid and the like.
Pharmaceutically acceptable salts also include base addition salts which may be formed when acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate and sodium hydroxide. Acceptable organic bases include diethanolamine, ethanolamine, N-methylglucamine, triethanolamine, trometharnine and the like.
xe2x80x9cSerine proteasexe2x80x9d means any enzyme that possesses a uniquely reactive serine residue that, through a mechanism of covalent attachment followed by hydrolysis, breaks amide bonds; thus, usually causing the degradation of proteins and peptides into smaller fragments. For the purposes of this invention, the definition of serine protease includes, but is not limited to protein C, chymase, chymotrypsin, cytomegalovirus protease, elastase, factor VIIa, factor IXa, factor Xa, plasm kallikrein, tissue kallikrein, xcex2-lactamase, plasmin, thrombin, trypsin, tryptase and urokinase.
xe2x80x9cSymptomatologyxe2x80x9d of a disease means any morbid phenomenon or departure from the normal in structure, function or sensation experienced by the patient and indicative of the disease, their production and the indications they finish.
xe2x80x9cTherapeutically effective amountxe2x80x9d means that amount which, when administered to an animal for treating a disease, is sufficient to effect such treatment for the disease.
xe2x80x9cTreatingxe2x80x9d or xe2x80x9ctreatmentxe2x80x9d of a disease includes preventing the disease from occurring in an animal which may be predisposed to the disease but does not yet experience or display symptoms of the disease, inhibiting the disease (i.e., arresting its development) or relieving the disease (i.e., causing regression of the disease).
Presently Preferred Embodiments
While the broadest definition of this invention is set forth in the Summary of the Invention, certain aspects of the invention are preferred. For example, preferred is a method for determining the serine protease inhibitory activity of a compound, which method comprises contacting the compound with a serine protease in a medium having present therein a divalent metal cation selected from the group consisting of zinc and cobalt, wherein the cation has the capacity for interaction with the compound and thereby to potentiate any serine protease inhibitory activity possessed by the compound and the concentration of the divalent metal cation in the medium is modified to a level sufficient to produce any such interaction; more preferably wherein the divalent metal cation is zinc.
Preferred methods of this invention are those in which an amount of zinc is present in the assay medium that is at least equal to the concentration of the serine protease inhibitor. Generally, the amount of zinc included in a preparation or medium containing the inhibitor will be at least 0.1 xcexcM, typically at least 1 xcexcM, more preferably at least 1 xcexcM, and more preferably at least 100 xcexcM. Typically, the zinc concentration employed will provide that at least about 80%, preferably at least about 90%, and more preferably substantially all of the inhibitor will be chelated with zinc. In physiological systems, the amount of zinc present will normally be sufficient to provide zinc complexes.
If the serine protease is sensitive to inhibition by the divalent metal cation, the method further may comprise contacting the compound with a serine protease in a medium having present therein a metal buffering agent capable of reducing the free concentration of the divalent metal cation to the extent that the serine protease is not substantially inhibited by the presence of the divalent metal cation while providing sufficient divalent metal cation by exchange equilibrium to produce the interaction. A typical metal buffer agent will have a Kd for chelation of the relevant divalent metal cation and will be present in suitable amounts such that the free concentration of the divalent metal cation is reduced to the extent that the cation is less available for association with the serine protease but the cation is available for association simultaneously with the protease and the protease inhibitor. Thus, the metal buffering agent will sequester all or most of the dissociated cation and readily transfer the cation to form a binary complex with the inhibitor or a ternary complex with inhibitor and protease (herein defined as exchange equilibrium). For example, a preferred method for determining the serine protease inhibitory activity of a compound wherein zinc is present in the medium and the serine protease is sensitive to inhibition by the zinc, further may comprise contacting the compound with a serine protease in a medium having present therein oxalate as the metal buffering agent.
Methods by which the concentration of the divalent metal cation can be modified and that fall within the intended scope of this invention include, but are not limited to, adding an appropriate amount of the divalent metal cation to the assay medium after the compound is contacted with the protease, mixing the assay reagents together with an appropriate amount of the divalent metal cation such that the desired final concentration of the divalent metal cation in the assay medium is achieved, selecting assay reagents by the levels of the divalent metal cation that are incidentally present so that after combining the reagents the final concentration of the divalent metal cation in the assay medium is achieved, and any other method that is applied for the purpose of adjusting the concentration in the assay medium such that it falls within the limitations of this invention.
Preferred is a method for determining whether the inhibitory activity of a serine protease inhibitor is enhanced by the presence of a divalent metal cation selected from the group consisting of zinc and cobalt, which method comprises:
(a) assaying for the inhibition of a serine protease by the inhibitor, wherein the assay is conducted in a medium that is essentially devoid of dissociated divalent metal cations; and
(b) assaying for the inhibition of the serine protease by the compound under essentially equivalent assay conditions to those used in Step (a), with the exception that the assay performed in Step (b) is conducted in a medium that contains an effective concentration of the divalent metal cation; wherein the inhibitory activity of the compound when measured by Step (b) is significantly greater than the inhibitory activity of the compound when measured by Step (a); more preferably wherein the divalent metal cations are removed from the medium used in Step (a) by the presence of a cation sequestering agent and most preferably wherein the divalent metal cation is zinc.
A typical cation sequestering agent will have a Kd for chelation of the relevant divalent metal cation such that the divalent metal cation is not available for association with the compound. For example, cation sequestering agents suitable in the practice of this invention include ethylenediaminetetraacetic acid (EDTA), phenanthroline, and any other common cation sequestering agent which has no effect on the protease activity on its own, preferably EDTA). Standard assay formats for the practice of this invention utilize the serine protease of choice and short peptide substrates whose cleavage can be monitored, typically by simple colorimetric methods. For the purposes of this invention, the inhibitory activity of a compound when measured by Step (b) is considered to be significantly greater than the inhibitory activity of the compound when measured by Step (a) if the difference in the activity is such that one of ordinary skill in the art would consider it substantive in view of typical biological and experimental variablity. Typically, a significantly greater activity will mean at least a ten, preferably at least a one hundred and more preferably at least a one thousand, fold increase in inhibitory activity.
Preferred is a method for inhibiting a serine protease with a divalent metal cation selected from the group consisting of zinc and cobalt in a medium comprising the serine protease and the inhibitor, in which the inhibitor comprises two heteroatoms in spatial relationship one to the other so as to chelate zinc, which method comprises having sufficient quantity of the divalent metal cation in the medium or adding a sufficient amount of the divalent metal cation to the medium to have any or all of the inhibitor which is bound to the serine protease as a divalent metal cation ternary complex, or providing the inhibitor as a divalent metal cation binary complex, such that the divalent metal cation is bound between the inhibitor and the active site of the serine protease as a divalent metal cation ternary complex; more preferably wherein the two heteroatoms are annular members of a ring, particularly wherein at least one of the heteroatoms is an annular member of an benzimidazole ring, or wherein the inhibitor is provided as a zinc binary complex. Suitable zinc binary complexes of compounds can be prepared by combining the compound with a zinc concentration of at least 0.01 xcexcM to 100 mM, typically 0.1 xcexcM to 50 mM and preferably about 5 xcexcM to 50 xcexcM.
Preferred methods of this invention for inhibiting a serine protease with a serine protease inhibitor are those in which the amount of zinc included in the assay medium is at least equal to the concentration of the serine protease inhibitor. Generally, the amount of zinc included in a preparation or medium containing the inhibitor will be at least 0.1 xcexcM, typically to least 1 xcexcM, more preferably 10 xcexcM, and more preferably at least 100 xcexcM. Typically, the zinc concentration employed will provide that at least about 80%, preferably at least about 90%, and more preferably substantially all of the inhibitor will be chelated with zinc. In physiological systems, the amount of zinc present will normally be sufficient to provide zinc complexes.
Preferred is a method for inhibiting a serine protease with a serine protease inhibitor comprising a bis(benzimidazole) in a medium comprising the serine protease and the inhibitor, wherein the inhibitor comprises nitrogen atoms of the bis(benzimidazole) in spatial relationship to chelate zinc, the improvement which comprises: adding sufficient zinc to the medium to have all of the inhibitor which is bound to the serine protease as a zinc complex or providing the inhibitor as a zinc binary complex; more preferably wherein the bis(benzimidazole) is substituted with an amidino group.
Preferred is a divalent metal cation binary complex of a compound of Formula II:
(Bp)r{xcex1-(A)-xcex2}xe2x80x83xe2x80x83II
in which:
BP is a binding moiety for binding to one or more P sites of a serine protease;
r is 0 or 1, with the proviso that at least one BP binding moiety is present;
A is a bond or a linking group selected from alkylene, heteroalkylene, cycloalkylene, arylene and heteroarylene, which linking group separates xcex1 and xcex2 by one to two atoms and is optionally substituted with 1 to 2 radicals selected from oxo, hydroxy, (C1-2)alkyloxy, halo, mercapto, (C1-2)alkylthio, amino, (C1-2)alkylamino and di(C1-2)alkylamino, wherein heteroatoms are bonded only to carbon or hydrogen; and
xcex1 and xcex2 each are independently a group comprised by two to thirty six carbon atoms, typically two to eighteen and more preferably two to twelve, and one to eight heteroatoms, wherein at least one heteroatom contained within each of xcex1 and xcex2 is within three, typically two and more preferably one, atoms of A and within six, typically four, atoms of the other heteroatom. Optionally xcex1 and xcex2 are linked via a linking group selected from alkylene and heteroalkylene, which group is optionally substituted with 1 to 2 radicals selected from oxo, hydroxy, (C1-2)alkyloxy, halo, mercapto, (C1-2)alkylthio, amino, (C1-2)alkylamino and di(C1-2)alkylamino, wherein heteroatoms are bonded only to carbon or hydrogen, in such a manner so that the two related heteroatoms are sterically positioned in spatial relationship one to the other so as to facilitate a bidentate chelation of the divalent metal cation.
More preferred is a compound of Formula II in which xcex1 and xcex2 are independently a group comprised by a heterocycloalkyl or heteroaryl moiety bonded directly or indirectly to A, or a group comprised by an alkyl, cycloalkyl, aryl, heterocycloalkyl or heteroaryl moiety substituted with a heteroatom containing moiety, wherein at least one annular or non-annular heteroatom contained within each of xcex1 and xcex2 is within three, typically two and more preferably one, atoms of A and within six, typically four, atoms of the other heteroatom.
Also preferred is a divalent metal cation binary complex of a compound of Formula III:
(BP){Xa-(xcex3)-Xb}xe2x80x83xe2x80x83III
in which:
BP is a binding moiety for binding to one or more P sites of a serine protease; and
xcex3 is an aromatic or non-aromatic, saturated or unsaturated, fused polycyclic hydrocarbon comprised by six to eighteen carbon atoms; and
Xa and Xb are independently an annular heteroatom contained within xcex3 or a heteroatom containing moiety bonded directly to xcex3, wherein Xa and Xb are within six, typically four, atoms of each other.
Representative divalent metal cations include cobalt and zinc. Zinc is one of the more common divalent metal cations present in physiological tissues and fluids, typically present at 10-100 xcexcM concentrations. Accordingly, preferred embodiments of this invention include zinc as the divalent metal cation.
Representative first BP binding moieties comprise a basic group bonded to the xcex1 or xcex2 group through a carbon atom, which basic group generally is comprised of carbon, hydrogen, nitrogen and/or oxygen, typically of not more than 10, preferably not more than 6, atoms other than hydrogen (e.g., guanidino, amidino, aminomethyl, amino higher alkyl, xcex1-aminocarboxymethyl, xcex1-aminocarboxamidemethyl, and the like). A representative second BP binding moiety generally is comprised of carbon, hydrogen, nitrogen and/or oxygen, typically of not more than 20 atoms other than hydrogen (e.g., alkyl, non-oxo carbonyl, amino, aminoalkyl, amidino, or the like). Further, a BP binding moiety may be an amino acid radical, particularly argininyl or lysinyl, may be an oligopeptide radical which is the specific target site for the target serine protease (e.g., amidino, amino, guanidino, or other basic moiety for trypsin-like serine proteases and carboxylates or lipophilic groups for chymotrypsin-like proteases).
Representative A linking groups include methylene, methene, carbonyl, amino, oxy, thio, isopropylidene, 1,2-cyclohexylene, 1,2-phenylene, and the like.
The xcex1 and/or xcex2 groups may have one or more heteroatom containing substituents other than the heteroatoms involved in chelating. The substituents will usually be not more than 6 carbon atoms, more usually not more than 3 carbon atoms and may include amino of from 0 to 6 carbon atoms, non-oxo-carbonyl of from 1 to 6 carbon atoms, particularly salts, esters and amides, and the sulfur and nitrogen analogs thereof, hydroxy or alkyloxy of from 0 to 6 carbon atoms, or aryloxy, halo, cyano, nitro, oxo, etc.
Representative xcex1 and/or xcex2 heteroaryl groups preferably have at least one sp2 nitrogen atom and include five membered rings, such as pyrazole, imidazole, triazole, oxazole, thiazole, isoxazole, etc. and benzo-fused derivatives thereof, such as benzimidazole, etc., six membered rings such as pyran, pyridine, pyridazine, pyrimidine, pyrazine, dioxane, etc. and benzo-fused derivatives thereof, such as quinoline, isoquinoline, cinnoline, etc., and non-fused rings, such as 2,2xe2x80x2-bis-imidazole, 2,2xe2x80x2-bis(pyridine), etc. The heteroaryl groups are optionally substituted with 1 to 3 radicals selected from halo, alkyloxy, amino, cyano, non-oxo-carbonyl, alkyl, or any other common substituent, preferably electron-donating, which do not sterically preclude binding or completion steps necessary for the inhibitor to function. Additional representative xcex1 and xcex2 groups include methoxymethyl, aminomethyl, methylaminomethyl, guanidinomethyl, amidinomethyl, anilinomethyl, 2,3-diaminopropyl, 2-amino-3-hydroxypropyl, 2-amino-2-trifluoromethylethyl, 2-hydroxyethyl, substituted aromatics like 2-mercaptophenyl, 2-hydroxyphenyl, 2-aminophenyl, 2-carboxyphenyl and substituted analogs thereof, methylacetoxy, glycinamidomethyl, cyclohexylaminomethyl, and the like.
Representative xcex3 groups include 1,8-naphthalenylene, 8-cinnolinyl, 5,6-phenanthrenylene, 1,8-dihydroxynaphthalenylene, and the like, wherein the atom(s) with free valence are bonded to heteroatom containing moiety.
In general, the compounds useful in the practice of this invention will comprise not more than sixty, typically not more than thirty six, carbon atoms and not more than twenty, typically not more than sixteen, preferably not more than twelve and more preferably not more than eight, heteroatoms. For example, representative serine protease inhibitors useful in the practice of this invention include 2,2xe2x80x2-bis(benzimidazoles) having one BP binding moiety at its 5-position and optionally having a second benzimidazolyl moiety in its 4xe2x80x2-or 5xe2x80x2-position.
In particular, compounds useful in the practice of this invention include, but are not limited to, bis(5-amidinobenzimidazol-2-yl)methane (Compound 1); bis(5-amidinobenzimidazole)-methanone (Compound 2); 2-(5-aminomethylbenzimidazol-2-ylmethyl)benzimidazole; 2-(5-amino-methylbenzimidazol-2-ylmethyl)-5-methylbenzimidazole (Compound 7); 2-(5-amidino-benzimidazol-2-ylmethyl)benzimidazole (Compound 4); 2-benzimidazol-2-ylethylbenzimidazole; 2-(5-guanidinobenzimidazol-2-ylmethyl)benzimidazole; 2-(5-carboxybenzimidazol-2-ylmethyl)-benzimidazole (Compound 8); 2-(imidazol-2-ylmethyl)-5-amidinobenzimidazole (Compound 6); 5-amidino-2-imidazol-4-ylmethylbenzimidazole; 5-amidino-2-pyrid-2-ylbenzimidazole (Compound 10); 5-amidino-2-pyrid-2-ylmethylbenzimidazole; 1-(5-amidinobenzimidazol-2-yl)-isoquinoline; 2-(5-amidinobenzimidazol-2-yl)quinoline; 3-(5-amnidinobenzimidazol-2-yl)-isoquinoline; 8-(5-amidinobenzimidazol-2-yl)quinoline; 5-amidino-2-(2-hydroxyphenyl)-benzimidazole; 5-amidino-2-(2-mercaptophenyl)benzimidazole; and 5-amidino-2-(2-aminophenyl)-benzimidazole.
The compounds useful in the practice of this invention can be prepared in accordance with known synthetic procedures. See, for example, Tidwell, et al., J. Med. Chem. (1978) 21:613-623; and general methods for the synthesis of substituted and/or fused heterocyclic systems and their isomers, as described in xe2x80x9cComprehensive Heterocyclic Chemistryxe2x80x9d, Pergamon Press:Oxford, 1988. The inhibitors may be prepared as crude mixtures comprising at least about 50 weight %, typically at least about 90, preferably at least 99, weight % of the composition.
Representative proteases useful in the practice of this invention include, but are not limited to, activated protein C, chymase, chymotrypsin, cytomegalovirus protease, elastase, elastase, factor VIIa, factor IXa, factor Xa, plasm kallikrein, tissue kallikrein, xcex2-lactamases, plasmin, thrombin, trypsin, tryptase and urokinase.
Pharmacology and Utility
The serine protease inhibitors useful in and identified by the practice of this invention, in association with,a divalent metal cation or as a pre-associated binary complex with a divalent metal cation, form a divalent metal cation ternary complex with the active site residues of the serine protease and thereby inhibits its activity. The serine protease inhibitors useful in and identified by the practice of this invention comprise (a) a chemical functional group which occupies the P1 site on the target serine protease and (b) a structurally adjacent bidentate chelator which captures the divalent cation into a tetrahedral complex involving side chains of His57 and Serl95 of the catalytic site of the enzyme. The combination of P1 binding and bidentate chelation properties in a single composition provides a synergistic effect for serine protease inhibition at physiological levels of divalent cations like zinc. For example, benzamidine (Compound 3) and benzylamine (Compound 5), which each have a typical P1 recognition element, are weak inhibitors of the serine protease trypsin. Similarly, 2-pyrid-2-ylbenzimidazole (Compound 9), which comprises a typical zinc sequestering element, produces no inhibition of trypsin. However, 5-amidino-2-pyrid-2-yl-benzimidazole (Compound 10), which comprises the structural combination of the typical P1 binding and zinc sequestering elements, is a potent inhibitor of trypsin. Thus, the serine protease inhibitors useful in and identified by the practice of this invention meet the above-described structural requirements if the value of the Ki (Zinc removed)  greater than  greater than Ki (Zinc added), indicating inhibition is potentiated in the presence of zinc. Accordingly, the methods and compositions of this invention are useful for in vitro and in vivo inhibition of serine proteases and for the screening for and the identification of serine protease inhibitors and for protecting peptides and proteins from proteolytic degradation.
X-ray crystallography of the inhibitor-zinc-serine protease complexes have and can be obtained using contemporary biophysical methodologies and commercial instrumentation. Such crystallographic data have and can be used to conclusively determine if a serine protease inhibitor has the structural requirements necessary for zinc potentiation of serine protease inhibition. An example of such an X-ray crystallographic determination is presented below.
Serine protease enzymes of interest include, but are not limited to, trypsin-like enzymes, such as trypsin, plasm kallikrein, tissue kallikrein, plasmin, thrombin and tryptase; chymotrypsin-like enzymes, including chymotrypsin, cathepsin G. and chymase; elastase-like enzymes, including neutrophil elastase and elastase; and carboxypeptidase-like enzymes. These enzymes play a role in apoptosis, blood pressure regulation, cancer, cardiovascular function, blood clotting, lysis, chemotaxis, development, digestion, fertilization, hormone processing, immune response, complement, infection: bacterial, viral and parasitic inflammation, mast cells, and other cells, neurologic, pain and protein secretion. Serine protease targets in medicine include for cardiovascular treatments: thrombin, factor Xa, factor VIIa, and chymase; for infectious diseases involving parasites, viruses and bacteria: cytomegalovirus protease and xcex2-lactamases, serine proteases specific for the pathogen; for bleeding, urokinase, activated protein C and tPA; for inflammation, tryptase, chymase, neutrophil elastase, plasm kallikrein and tissue kallikrein; and for neurobiology, serine proteases associated with Alzheimer""s disease, to name only a few of the available targets.
The methods and compositions of this invention may be used in affinity columns to isolate serine proteases. Furthermore, because the serine protease inhibitors useful in the practice of this invention can vary as to their specificity toward serine proteases, the methods and compositions of this invention can be used to selectively isolate and purify serine proteases. In this regard, conventional techniques may be employed for linking the various compounds to supports, beads, macromolecules, and the like. Linking groups may include carboxyl groups, amino groups, thio groups, activated olefins, or the like. The linking group may be bonded to the inhibition moiety or the binding moiety. Surfaces of columns or capillaries may be employed as the affinity column or the column may be packed with a variety of beads, such as Sephadex, sepharose, latex beads, or the like. The particular manner in which the column is prepared is not critical to the practice to this invention. In addition, the columns may be used in assays for detecting the various serine proteases, by providing a binding profile which can be developed for the serine protease of interest, and determining relative Ki values for libraries of inhibitors that utilize metal complexation as a mechanism of inhibition.
The serine protease inhibitors useful in and identified by the practice of this invention are themselves useful for the treatment of various diseases. For example, serine protease inhibitors can be used for the treatment of clinical arthritis, synovitis and associated pathologies (e.g. see U.S. Pat. No. 4,940,723). The serine proteases useful in and identified by the practice of this invention also permit investigation of the physiological processes associated with a wide variety of biological events involving serine proteases. The advantage of being able to modulate serine protease activity by removing or adding zinc allows for the investigation on the effect of such activity in physiological processes.
Further, because the divalent metal cation-serine protease inhibitor complexes of this invention are able to form the inhibitor-cation-serine protease ternary complex without the necessity of having the concentration of zinc in the assay medium modified, the pre-associated cation-inhibitor complexes are useful in the practice the methods of this invention for identifying the inhibitory activity of the inhibitors. Similarly, the divalent metal cation-serine protease inhibitor complexes of this invention are useful in treating disease wherein the inhibitor is delivered to the target tissue locally (e.g., by topical application to the skin or eyes, by aerosol delivery to the lungs, etc.).
Administration and Pharmaceutical Compositions
In general, the divalent metal cation-serine protease inhibitor complexes of this invention and the serine protease inhibitors useful in and identified by the practice of this invention will be administered in therapeutically effective amounts via any of the usual and acceptable modes known in the art, either singly or in combination with another therapeutic agent. A therapeutically effective amount may vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compound used and other factors. For example, therapeutically effective amounts of a serine protease inhibitor or complexes thereof for the treatment of asthma may range from 0.1 micrograms per kilogram body weight (xcexcg/kg) per day to 1 milligram per kilogram body weight (mg/kg) per day, typically 1 xcexcg/kg/day to 0.1 mg/kg/day. Therefore, a therapeutically effective amount for a 80 kg asthmatic human patient may range from 10 xcexcg/day to 10 mg/day, typically 0.1 mg/day to 10 mg/day. In general, one of ordinary skill in the art, acting in reliance upon personal knowledge and the disclosure of this application, will be able to ascertain the therapeutically effective amounts of the serine protease inhibitors for treating a given disease.
Serine protease inhibitors can be administered as pharmaceutical compositions by one of the following routes: oral, systemic (e.g., transdermal, intranasal or by suppository) or parenteral (e.g., intramuscular, intravenous or subcutaneous). Compositions can take the form of tablets, pills, capsules, semisolids, powders, sustained release formulations, solutions, suspensions, elixirs, aerosols, or any other appropriate composition and are comprised of, in general, a serine protease inhibitor in combination with at least one pharmaceutically acceptable excipient. Acceptable excipients are non-toxic, aid administration, and do not adversely affect the therapeutic benefit of the active ingredient. Such excipient may be any solid, liquid, semisolid or, in the case of an aerosol composition, gaseous excipient that is generally available to one of skill in the art.
Solid pharmaceutical excipients include starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk, and the like. Liquid and semisolid excipients may be selected from water, ethanol, glycerol, propylene glycol and various oils, including those of petroleum, animal, vegetable or synthetic origin (e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc.). Preferred liquid carriers, particularly for injectable solutions, include water, saline, aqueous dextrose and glycols.
Compressed gases may be used to disperse the active ingredient in aerosol form. Inert gases suitable for this purpose are nitrogen, carbon dioxide, nitrous oxide, etc. Other suitable pharmaceutical carriers and their formulations are described in A. R. Alfonso Remington""s Pharmaceutical Sciences 1985, 17th ed. Easton, Pa.: Mack Publishing Company.
The amount of a serine protease inhibitor or metal cation complex thereof in the composition may vary widely depending upon the type of formulation, size of a unit dosage, kind of excipients and other factors known to those of skill in the art of pharmaceutical sciences. In general, a composition of a serine protease or metal cation complex thereof will comprise from 0.01%w to 10% w, preferably 0.3% w to 1% w, of active ingredient with the remainder being the excipient or excipients. Preferably the pharmaceutical composition is administered in a single unit dosage form for continuous treatment or in a single unit dosage form ad libitum when relief of symptoms is specifically required.